RESUMO
Background: Baculovirus expression system is one of the most attractive and powerful eukaryotic expression systems for the production of recombinant proteins. The presence of a biomarker is required to monitor transfection efficiency or protein expression levels in insect cells
Methods: The aim of this study was to construct a baculovirus expression vector encoding a copepod super green fluorescent protein [copGFP]. In this light, the resultant vector was constructed and used for transfection of Spodoptera frugiperda cells
Results: Expression of the copGFP protein in insect cells was confirmed by fluorescent microscopy and Western-blot analysis
Conclusion: The application of copGFP control bacmid can be considered as an appropriate control for insect cell transfection
RESUMO
Background: During the recent years, significant progress has been achieved on development of novel anti-viral drugs. Natural products are assumed as the potential sources of novel anti-viral drugs; therefore, there are some previous studies reporting the anti-viral compounds from venomous animals. Based on the significant value for tracing of non-toxic anti-viral agents from natural resources, this study was aimed to investigate the anti-viral activity of some HPLC purified fractions derived from the venom of Iranian scorpion, Hemiscorpius lepturus, against human immunodeficiency virus 1 [HIV-1] and herpes simplex virus 1 [HSV-1]
Methods: H. Lepturus crude venom was subjected to reverse phase HPLC analysis to determine its active components precisely where four dominant fractions obtained at retention time of 156-160 minutes. The phospholipase A2 and hemolytic activities of the purified fractions were first evaluated. Then the anti-viral activity was measured using single cycle HIV [NL4-3] replication and HSV [KOS] plaque reduction assays
Results: The H. lepturus crude venom inhibited HIV replication by 73% at the concentration of 200 micro g/ml, while it did not show significant anti-HSV activity. It also inhibited the cell-free viral particles in a virucidal assay, while it showed no toxicity for the target cells in a proliferation assay. The four HPLC fractions purified from H. lepturus inhibited HIV with IC50 of 20 micro g/ml
Conclusion: H. lepturus venom contains components with considerable anti-HIV activity insofar as it has virucidal activity that offers a novel therapeutic approach against HIV infection. Our results suggest a promising pilot for anti-HIV drug discovery with H. lepturus scorpion venom
RESUMO
In this study we co-administered melittin along with HBsAg/alum vaccine to investigate if it helps elicitation of Th1/Th2 response. Hepatitis B virus [HBV] infection is a life-threatening liver infection, which can lead to chronic liver disease. Vigorous T cell responses are stimulated at acute, self-limiting HBV infection, while chronic HBV infection elicits very weak T cell responses. The prevalence of HBV infection has been decreased by the approved vaccination approach using recombinant HBs antigen [HBsAg] and alum i.e. HBV vaccine. Alum, a strong Th2 stimulator, is usually used as adjuvant to increase HBsAg immunogenicity. The present vaccine does not induce protective and/or prophylactic immune response in some groups. Melittin, major active component in the venom of honeybee, induces Th1 development. Experimental mice were immunized with melittin plus hepatitis B vaccine on day 0 following by two booster doses with the same injections. Lymphocyte proliferation, IFN-gamma, and IL-4 level, total antibody and isotyping of IgG1, IgG2a IgG2b, and IgM were measured using ELISA. Administration of melittin and HBV vaccine had no effect on lymphoproliferation and total antibody responses, but increased IFN-gamma response and induced Th1 response. The present study proposed that administration of melittin along with conventional vaccine shifts T cell responses towards Th1/Th2 dominated with Th1 response. The resultant immune response leads to activation of both cell-mediated and humoral immune responses, both of which required for clearance of HBV infection.
RESUMO
Staphylococcus aureus is a leading cause of many nosocomial and community acquired infections. According to many reports, antibiotic therapy cannot guarantee the eradication of S. aureus infections. Thus designing an adhesin based vaccine could restrain the S. aureus infections. This study designed for construction of a new fusion protein vaccine against S. aureus infections based on adhesin molecules fibronectin binding protein A [FnBPA] and clumping factor A [ClfA]. Bioinformatic experiments were performed using Oligo analyzer and DNAMAN softwares. The fragments corresponding to fnbA binding domain and a C-terminal fragment from clfA were amplified from S. aureus NCTC8325 genomic DNA. Purified PCR products and the vector, pET15b, were digested with NcoI and BamHI. The digested PCR products were hybridized together and then ligated to digested vector. Finally incomplete construct was assembled by Taq DNA polymerase. To quick confirmation of cloning procedure the new construct designated pfnbA-clfA was digested with NcoI and BamHI. To further verification, the product was sent for sequencing. The data based on bioinformatics analysis showed no homology between fusion protein and human proteins. Digestion of new vector with NcoI and BamHI confirmed the ligation of fusion protein sequence into pET15b. Sequencing results verified the integrity of target sequences. This study is the first effort to construct a new fusion protein vector based on S. aureus adhesins using a new design. This project is being continued to study the expression and biological activity of the fusion protein in a cell culture model